https://onlinelibrary.wiley.com/doi/pdf/10.1021/js950534b

Pharmaceutical Applications of Cyclodextrins. 1. Drug Solubilization and Stabilization

Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.

Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability

Lyophilization and development of solid protein pharmaceuticals

Irreversible protein aggregation is problematic in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage processes. The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.

Physical Stability of Proteins in Aqueous Solution: Mechanism and Driving Forces in Nonnative Protein Aggregation

Instability, stabilization, and formulation of liquid protein pharmaceuticals.

Recombinant DNA technology has now made it possible to produce proteins for pharmaceutical applications Consequently, proteins produced via biotechnology now comprise a significant portion of the drugs currently under development Isolation, purification, formulation, and delivery of proteins represent significant challenges to pharmaceutical scientists, as proteins possess unique chemical and physical properties These properties pose difficult stability problems A summary of both chemical and physical decomposition pathways for proteins is given Chemical instability can include proteolysis, deamidation, oxidation, racemization, and β-elimination Physical instability refers to processes such as aggregation, precipitation, denaturation, and adsorption to surfaces Current methodology to stabilize proteins is presented, including additives, excipients, chemical modification, and the use of site-directed mutagenesis to produce a more stable protein species

Stability of Protein Pharmaceuticals

Deamidation of Asn residues is a major chemical pathway of degradation of peptides and proteins. To understand better the external factors that influence deamidation, we studied the degradation of the hexapeptide Val–Tyr–Pro–Asn–Gly–Ala, a fragment of adrenocorticotropic hormone, by HPLC. The deamidation of this model peptide showed marked dependence on pH, temperature, and buffer composition. In the pH range 5 to 12, the peptide deamidated exclusively via a cyclic imide intermediate with the formation of both the Asp- and the isoAsp-hexapeptides. Buffer catalysis was also observed in the pH range of 7 to 11. However, at acidic pH's, the pathway of deamidation involved direct hydrolysis of the amide side chain of Asn residue to produce only the Asp-hexapeptide.

Chemical Pathways of Peptide Degradation. II. Kinetics of Deamidation of an Asparaginyl Residue in a Model Hexapeptide

Deamidation of Asn residues can occur either by direct hydrolysis of the Asn residue or via a cyclic imide intermediate. The effects of primary sequence on the pathways of deamidation of Asn residues were studied using Val-Tyr-X-Asn-Y-Ala hexapeptides with substitution on the C-terminal side (Y) and on the N-terminal side (X) of the Asn residue. In acidic media the peptides deamidate by direct hydrolysis of the Asn residue to yield only Asp peptides, whereas under neutral or alkaline conditions, the peptides deamidate by formation of the cyclic imide intermediates which hydrolyze to yield both isoAsp and Asp peptides. At neutral to alkaline pH's the rate of deamidation was significantly affected by the size of the amino acid on the C-terminal side of the Asn residue. The amino acid on the C-terminal side of the Asn residue has no effect on the rate of deamidation at acidic pH. Changes in the structure of the amino acid on the N-terminal side of the Asn residue had no significant effect on the rate of deamidation at all the pH's studied. For peptides that underwent deamidation slowly, a reaction involving the attack of the Asn side chain on the peptide carbonyl carbon resulting in peptide bond cleavage was also observed.

Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides.

Deamidation of Asn residues is one of the major chemical pathways of degradation of proteins and peptides. Adrenocorticotropic hormone (ACTH), a 39-amino acid polypeptide with a single Asn residue, was shown in this study to be a useful model polypeptide for the study of the effects of pH and buffer concentration on the rate and pathway of deamidation. The disappearance of ACTH and appearance of deamidated ACTH were monitored by isoelectric focusing (IEF), and ammonia production was monitored spectrophotometrically using a coupled enzymatic assay. Using these analytical methods, the deamidation of ACTH was shown to follow pseudo-first-order kinetics and was dependent on pH and buffer concentrations. The separation of the deamidated ACTHs (Asp-ACTH and isoAsp-ACTH) from ACTH was successful, but attempts to separate Asp-ACTH from isoAsp-ACTH using cation-exchange HPLC and IEF were unsuccessful. Using bovine protein carboxymethyltransferase (PCM), which selectively methylates the carboxyl group of isoAsp residue, the isoAsp-ACTH could be detected at pH 7.0 and 9.6 but not at pH 1.9. These data support the hypothesis that under neutral and alkaline conditions, deamidation of ACTH proceeds through the formation of a cyclic imide intermediate (slow step), followed by its hydrolysis to the Asp-ACTH and isoAsp-ACTH (fast step). Under acidic conditions, the reaction appears to proceed via direct hydrolysis of the Asn residue to form Asp-ACTH without the formation of a cyclic imide intermediate.

Kamlesh Patel

Papers

Stability of Protein Pharmaceuticals

Chemical Pathways of Peptide Degradation. II. Kinetics of Deamidation of an Asparaginyl Residue in a Model Hexapeptide

Chemical pathways of peptide degradation. III. Effect of primary sequence on the pathways of deamidation of asparaginyl residues in hexapeptides.

Chemical pathways of peptide degradation. I. Deamidation of adrenocorticotropic hormone.

Deamidation of asparaginyl residues in proteins: a potential pathway for chemical degradation of proteins in lyophilized dosage forms.